AKT2S128/CCTαS315/319/323-positive cancer-associated fibroblasts (CAFs) mediate focal adhesion kinase (FAK) inhibitors resistance via secreting phosphatidylcholines (PCs)

Abnormal metabolism is regarded as an oncogenic hallmark related to tumor progression and therapeutic resistance. Present study employed multi-omics, including phosphoproteomics, untargeted metabolomics and lipidomics, to demonstrate that the pAKT2 Ser128 and pCCTα Ser315/319/323-positive cancer-associated fibroblasts (CAFs) substantially release phosphatidylcholines (PCs), contributing to the resistance of focal adhesion kinase (FAK) inhibitors in esophageal squamous cell carcinoma (ESCC) treatment. Additionally, we observed extremely low levels of FAK Tyr397 expression in CAFs, potentially offering no available target for FAK inhibitors playing their anti-growth role in CAFs. Consequently, FAK inhibitor increased the intracellular concentration of Ca2+ in CAFs, promoting the formation of AKT2/CCTα complex, leading to phosphorylation of CCTα Ser315/319/323 sites and eventually enhancing stromal PC production. This activation could stimulate the intratumoral Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (STAT3) pathway, triggering resistance to FAK inhibition. Analysis of clinical samples demonstrated that stromal pAKT2 Ser128 and pCCTα Ser315/319/323 are related to the tumor malignancy and reduced patient survival. Pseudo-targeted lipidomics and further validation cohort quantitatively showed that plasma PCs enable to distinguish the malignant extent of ESCC patients. In conclusion, inhibition of stroma-derived PCs and related pathway could be possible therapeutic strategies for tumor therapy.


INTRODUCTION
2][3] Various risk factors, such as tobacco or alcohol addiction, genetic defects, and some other detrimental environmental factors may possibly induce the formation and development of ESCC.Importantly, the poor survival rate is resulted from the shortage for therapeutic efficacy from cytotoxic, targeted and immune-based therapeutics. 4,5Integrated multi-omics analysis of ESCC will yield precise molecular classification for exploring new diagnostics markers and therapeutic targets and then enhance the efficacy of ESCC treatment.Correspondingly, exploration of signaling addiction, vulnerability, or some other important tumor-related pathways and evaluation of their targetability and druggability can provide research paradigm for precision therapy against ESCC.
7][8] CAFs play the central role in the TME of solid tumors to induce various malignant phenotypes of tumors, including persistent growth, invasion and metastasis, angiogenesis, epithelial-mesenchymal transition (EMT), and the formation of tumor stem cells.3][14] The alteration of metabolites and their relevant intermediates effectively rewire tumor cells and the cellular components of TME to boost the output of lipid, protein, glucose and other important metabolism-related pathways.Furthermore, the expression changes or genetic mutations of key metabolic enzymes in tumor cells and their surrounding TME can dramatically elevate the concentration of metabolites in tumor cells and TME, and subsequently reshape TME and reprogram tumor cells to support the tumor malignancy and induce chemotherapy resistance.How tumor cells utilize metabolic nutrients and their affected signaling pathways are filed of concentrative investigation.With the purpose of managing the metabolic challenges imposed by the TME, tumor cells and CAFs cooperatively interact to facilitate tumor malignancy.Moreover, it remains unclear what are the metabolic profiles of CAFs and how CAFs-derived metabolites act on tumor malignancy and the response of tumor cells towards therapeutic agents.
Dysregulation of tumor-promoting kinases' activities has been focused, due to tumor cells can utilize these proteins to enhance the tumor cells/TME interaction, and evade immune surveillance and then induce the malignant progression or metastasis of tumor cells.Selection of kinase targets and evaluation of their antitumor efficacy and related molecular mechanisms are critical for the development of antitumor agents.Focal adhesion kinase (FAK) refers to the cytoplasmic non-receptor protein tyrosine kinase and can be ubiquitously expressed. 15,167][18][19][20] Under the stimulation of signalings from tumor cells themselves and cellular components of TME, intratumoral FAK can facilitate many cellular or biological activities or reactions of tumor cells via its kinasedependent function, or act as scaffolding protein to influence the assembly of several protein signalosomes and resultantly promote the uncontrollable growth and sustained invasion and metastasis of tumor cells.These findings have contributed to developing FAK inhibitors for the clinical treatment of tumors.Many phase I or II clinical trials have been approved or conducted to observe the efficacy of FAK inhibitor alone or in combination with other antitumor agents in tumor treatment.Nevertheless, the clinical effect of FAK inhibitors remains controversial, even though some FAK inhibitors have made satisfying antitumor impact on preclinical studies only with in vitro assays. 15,21It can be hypothesized that this discrepancy is at least in part triggered by CAFs, secreting some substances to promote the dysregulation of intratumoral signaling pathways, as well as resultantly impairing the antitumor efficacy of chemotherapies.
While several studies have explored cytokines, chemokines or some growth factors secreted by tumor cells or CAFs mediate the crosstalk between these two types of cells.The in-depth understanding of TME-derived metabolites which regulate tumor and CAFs communications in therapeutic resistance of tumor cells still needs to be explored.In this work, we comprehensively exploited the ESCC CAFs-derived metabolic profiles and aimed at investigating whether CAFs-derived metabolites can be applied as biomarkers to identify the progression of tumor malignancy and how these metabolites change the antitumor effect of FAK inhibitors via the regulation of the intercellular signaling crosstalk between tumor cell and CAFs.
To assess whether CAFs contribute to the resistance of FAK inhibitor in ESCC treatment in vivo, KYSE410 or KYSE510 cells and CAFs #1 or #5 were respectively coinjected into BALB/c-nu mice.1d).CAFs #5 could not affect defactinib-mediated inhibition of tumor growth in indicated ESCC cells/CAFs #5 coinjection xenografted model (Fig. 1e).
To comprehensively evaluate the antitumor effect of defactinib in ESCC/CAFs coinjection xenografted model, the expression of Ki-67, CD31, and LYVE-1 in tumor tissues was measured with quantitative ELISA assays.As the results of Supplementary Fig. 2a-f shown, defactinib inhibited the expression of Ki-67, CD31 and LYVE-1 in KYSE410 and KYSE510 tumors (Supplementary Fig. 2a-c), but could not suppress the expression of these biomarkers in KYSE410 and KYSE510 tumors in the presence of CAFs #1 (Supplementary Fig. 2d-f).However, defactinib decreased the level of Ki-67, CD31 and LYVE-1 in KYSE410 or KYSE510 tumors/ CAFs #5 (Supplementary Fig. 2g-i).
3][24] We have coinjected KYSE410 or KYSE510 cells/CAFs #1 or #5 into the footpads of mice, and then observed the anti-metastatic ability of defactinib in this xenografted model.As the results in Supplementary Fig. 3a shown, defactinib could not effectively inhibit the CAFs #1-facilitated formation of larger lymph nodes of KYSE410 and KYSE510 tumors.However, CAFs #5 could not affect the antimetastatic ability of defactinib in the in vivo lymph node metastasis model (Supplementary Fig. 3b).
We chose two PCs-PC (16:0/20:4) and glycerophosphocholine for further functional assays to evaluate whether PCs induce the malignant progression of tumor cells, and found that these two PCs (10 μM) effectively stimulated the growth and invasion of KYSE410 and KYSE510 cells (Fig. 2g, h).CTP-phosphocholine cytidyltransferase (CCT) enzymes (including CCTα and β) catalyze the key rate-limiting step in choline pathway for phosphatidylcholine (PC) biosynthesis.We then knocked down CCTα and β in CAFs using siRNA, and further observed whether CCTα or β-depleted CAFs can contribute to the malignancy of ESCC cells.Depletion of CCTα in CAFs effectively blocked the secretion of PCs from CAFs (Supplementary Fig. 6a, b).Correspondingly, CCTα siRNAs impaired CAFs-induced the growth and invasion of KYSE410 and KYSE510 cells in ESCC cells/CAFs coculture system (Supplementary Fig. 6c, d).Furthermore, CCT inhibitor-miltefosine (25 μM) effectively inhibited the CAFs-induced ESCC malignancy (Supplementary Fig. 6e, f).
Phosphoproteomics identifies that the activation of AKT2/CCTα axis in CAFs contributes to the secretion of PCs We further explored the molecular mechanism of the secretion of PCs from CAFs using phosphoproteomics.Interestingly, AKT2 Ser 128 was appeared in almost all of these identified pathways in CAFs #1 treated with defactinib (10 μM), and the upregulated CCTα Ser 315/319/323 sites existed in choline metabolism in cancer (KEGG ID: hsa05231) (Fig. 4a).
We found that AKT2 and CCTα and their phosphorylated forms-AKT2 Ser 128 and CCTα Ser 315/319/323 were highly expressed in CAFs #1-4 (AKT2 S128 /CCTα S315/319/323 -positive CAFs), whereas not in CAFs #5 (AKT2 S128 /CCTα S315/319/323 -negative CAFs) (Fig. 4b).We Fig. 1 CAFs impairs the antitumor effect of defactinib.a, b Transwell apparatus with 0.4 μm pore size was used to evaluate the tumor growth inhibitory effect of defactinib and VS4718, the CAFs #1-#5 were respectively plated in the upper chamber of transwell plates.The KYSE410 or KYSE510 cells were respectively plated in the lower chamber of transwell plates (a).After cells were seeded, defactinib or VS4718 (0-10 μM) was added, incubated for 4 days, and then growth of indicated ESCC cells was measured using MTS assay.IC 50 value of defactinib (left panel) or VS4718 (right panel) in KYSE410 and KYSE510 cells was shown.***P < 0.001 as IC 50 value of defactinib or VS4718 in KYSE410 and KYSE510 cells cultured alone compared with that of defactinib or VS4718 in KYSE410 and KYSE510 cells/CAFs #1 to #4 coculture system.### P < 0.001 as IC 50 value of defactinib or VS4718 in KYSE410 and KYSE510 cells/CAFs #1 to #4 coculture system compared with that of defactinib or VS4718 in KYSE410 and KYSE510 cells/CAFs #5 coculture system (b).c CAFs #1-#5 were treated with defactinib or VS4718 (0-25 μM) for 4 days, and the cell growth was evaluated using MTS assay.IC 50 value of defactinib or VS4718 in CAFs was shown.Error bars, mean ± SD of three independent experiments.d KYSE410 (left panel) or KYSE510 (right panel) cells were respectively coinjected with CAFs #1 into the flank of BALB/c mouse.After the xenografts reached at approximately 80-100 mm 3 .Tumor cells with/without CAFs #1 were treated with control vehicle or defactinib (25 mg/kg/day, p.o.), respectively.Tumor volume was measured every 4 days for the indicated period.Curves of tumor volume were listed.e The experimental protocol of e was similar with that of d except the CAFs were chosen CAFs #5.n.s.no significant difference; ***P < 0.001.Error bars, mean ± SD of five independent experiments.f Transwell apparatus with 8 μm pore size was used to evaluate the anti-invasive ability of defactinib and VS4718, the CAFs #1-#5 were respectively plated in the lower chamber of transwell plates.The KYSE410 or KYSE510 cells were respectively plated in the upper chamber of transwell plates.After cells were seeded, 10 μM defactinib (g: in KYSE410 cells; i: in KYSE510 cells) or VS4718 (h: in KYSE410 cells; j: in KYSE510 cells) was added, incubated for 24 h, and then invasion of ESCC cells was measured using transwell assay.The invasive ratio of defactinib or VS4718/respective control was listed.***P < 0.001 as the invasive ratio of defactinib or VS4718 in KYSE410 or KYSE510 cells cultured alone compared with that of defactinib or VS4718 in KYSE410 and KYSE510 cells/CAFs #1 to #4 coculture system.### P < 0.001 as the invasive ratio of defactinib or VS4718 in KYSE410 and KYSE510 cells/CAFs #1 to #4 coculture system compared with that of defactinib or VS4718 in KYSE410 and KYSE510 cells/CAFs #5 coculture system c The defactinib (10 μM) upregulated representative metabolites were shown using heatmap.d The experimental condition of d is consistent with a.The CM from CAFs #1 was subjected to lipidomics.The 10 enriched pathways were shown using bubble chart.Levels of PCs secreted from e 5 cases of CAFs, 4 cases of TAMs, 3 cases of ECs, 2 cases of ESCC and ESCC cell lines-KYSE410 and KYSE510 or f 5 cases of CAFs with/ without 10 μM defactinib or VS4718, were evaluated using quantitative PCs ELISA assay.g, h KYSE410 and KYSE510 cells were treated with 10 μM PC (16:0/20:4) or glycerophospholipid for 4 days, then the growth of indicated ESCC cells was evaluated using MTS assay (g); or for 24 h, the invasion of indicated ESCC cells was evaluated using Transwell assay (h).***P < 0.001.Error bars, mean ± SD of five independent experiments assessed whether FAK inhibition affected the phosphorylation of AKT2 Ser 128 and CCTα Ser 315/319/323 sites in CAFs using coculture system (transwell apparatus with 0.4 μm pore size).The upper chamber of transwell apparatus was plated with KYSE410 or KYSE510 cells, and the lower chamber was cultured with CAFs.After 24 h defactinib treatment, the lysates of CAFs were collected for evaluation of the phosphorylation status of AKT2 Ser 128 and CCTα Ser 315/319/323 sites.Defactinib (10 μM) effectively stimulated the phosphorylation of AKT2 Ser 128 and CCTα Ser 315/319/323 sites in CAFs #1-4 alone or in the presence of KYSE410 or KYSE510 cells (Fig. 4c).However, defactinib (10 μM) could not induce the expression of AKT2 and CCTα and their indicated phosphorylation status in CAFs #5 alone or cocultured with ESCC cells (Fig. 4c).Function-loss AKT2 (S128A) was stably transfected into CAFs #1, c KYSE410 or KYSE510 cells were plated in the upper chamber of transwell plates with 0.4 μm pore size.The CAFs #1-#5 were plated in the lower chamber of transwell plates, and cocultured with/without 10 μM defactinib for 24 h.Then, lysates of CAFs #1-#5 were collected and subjected to immunoblotting assay for evaluating the expression of pCCTα Ser 315/319/323 , CCTα, pAKT2 Ser 128 , or AKT2.d CAFs #1 were stably transfected with control vector, loss-of-function AKT2 S128A plasmid, and the transfection efficacy was evaluated using immunoblotting to detect the expression of Flag.GAPDH was used as the loading control.e CAFs #1 harbored vector or loss-of-function AKT2 (S128A) plasmid were treated with/without defactinib (10 μM) for 24 h.The expression of pCCTα Ser 315/319/323 and CCTα was evaluated using immunoblotting assay.f The experimental condition of (f) was similar with that of c.Then, lysates of CAFs #1 were immunoprecipitated with AKT2 (IP: AKT2).Immunocomplexes were subsequently immunoblotted using AKT2 (IB: AKT2), pCCTα Ser 315/319/323 (IB: pCCTα Ser 315/319/323 ) or CCTα (IB: CCTα) antibody.g Defactinib (10 μM)-upregulated the phosphorylation of several Ca 2+ -related proteins and their located pathways were shown.h CAFs #1 were treated with defactinib (10 μM), and the concentration of intracellular Ca 2+ was quantified.i-k CAFs #1 were treated with defactinib (10 μM) in the presence or absence of Ca 2+ chelator-BAPTA-AM (10 μM).The secreted PCs was evaluated using quantitative PCs ELISA assay (i).The expression of pCCTα Ser 315/319/323 , CCTα, pAKT2 Ser 128 , or AKT2 was measured using immunoblotting assay (j).The interaction between AKT2 and CCTα was evaluated using IP-IB assay.Lysates of CAFs #1 were immunoprecipitated with AKT2 (IP: AKT2).Immunocomplexes were subsequently immunoblotted using AKT2 (IB: AKT2), pCCTα Ser 315/319/323 (IB: pCCTα Ser 315/319/323 ) or CCTα (IB: CCTα) antibody (k).***P < 0.001.Error bars, mean ± SD of five independent experiments and then the effect of FAK inhibition on the phosphorylation of CCTα Ser 315/319/323 was measured.Defactinib (10 μM) could not stimulate the phosphorylation of CCTα Ser 315/319/323 sites in CAFs harbored AKT2 S128A mutant (Fig. 4d, e).Correspondingly, defactinib (10 μM) could not induce the secretion of PCs from CAFs #1 stably transfected with AKT2 S128A or CCTα S315/319/ 323A mutant (Fig. 4d, Supplementary Fig. 10a, b).
We then analyzed whether defectinib affects the phosphorylation of AKT2 and CCTα in ESCC cells.CAFs were cultured in the upper chamber of transwell apparatus (0.4 μm pore size), and the KYSE410 or KYSE510 cells were respectively cultured in the lower chamber of transwell apparatus.After 24 h defactinib treatment, the lysates of indicated ESCC cells were collected for assessing the phosphorylation status of AKT2 Ser 128 and CCTα Ser 315/319/323 sites.Defactinib (10 μM) inhibited the phosphorylation of AKT2 Ser 128 and CCTα Ser 315/319/323 sites in KYSE410 or KYSE510 cells with or without CAFs #1 (Supplementary Fig. 11).

CAFs-released PCs activate intratumoral STAT3 to mediate the resistance of defactinib in ESCC treatment
Because intratumoral STAT3 contributed to the resistance of FAK inhibitor in tumor treatment. 28We evaluated whether defactinib stimulated the activation of intratumoral STAT3 in ESCC cells/CAFs coculture system, and found that defactinib (10 μM) upregulated the phosphorylation of STAT3 Tyr 705 in KYSE410 and KYSE510 cells in the presence of CAFs #1, compared with defactinib in KYSE410 or KYSE510 cells cultured alone (Fig. 6a).We then determined whether PCs induce the activation of intratumoral STAT3, and found that PC (16:0/20:4) and glycerophosphocholine (10 μM) increased the phosphorylation of STAT3 Tyr 705 in KYSE410 and KYSE510 cells (Supplementary Fig. 13a).Furthermore, AKT2 S128A, CCTα S315/319/323A mutant or CCTα siRNA effectively blocked CAFs (in the presence of defactinib)-induced intratumoral STAT3 activation in ESCC cells/CAFs #1 coculture system (Fig. 6a, b).Formation of Tyk2/JAK2 heterodimer is critical for persistent activation of intratumoral STAT3 and the resistance of targeted therapy, including ESCC cells. 24,29We evaluated whether PCs could stimulate the interaction between Tyk2 and JAK2 in ESCC cells, and found that PC (16:0/20:4) and glycerophosphocholine (10 μM) facilitated the formation of Tyk2/JAK2 complex and the activation of JAK2 in this complex (Supplementary Fig. 13b).
Our previous study demonstrated that the expression of pFAK Tyr 397 in ESCC tissues positively correlated with tumor malignancy. 30However, the staining intensity of pFAK Tyr 397 was low in stroma (22.2% high expression of pFAK Tyr 397 ; 24/108) (Fig. 7g).

DISCUSSION
In the present study, we show that FAK inhibitors stimulate AKT2 S128 /CCTα S315/319/323 -positive CAFs subset to secrete PCs, which induce malignant cells STAT3 activation to facilitate the therapeutic resistance of tumor cells.Present data establish a concept in CAFs-FAK-regulated and metabolites-mediated control of tumor malignancy with relevance to human ESCC with low stromal FAK expression, and detect potential novel actionable targets for anticancer therapy.Importantly, we found that plasma PCs can be served as biomarkers for classifying ESCC stage.
Our data show that FAK suppression increases the stromal level of PCs and their metabolites-LPCs, the major membrane structural phospholipids, and the stromal levels of other types of phospholipids, such as PE, PS, PI, or LPS.Moreover, FAK inhibition caused upregulation of unusual lipid subclass-the (O-acyl)-ωhydroxy FAs (OAHFAs) from CAFs, suggesting that inhibiting FAK activity results in disruption of stromal choline and its related glycerophospholipid homeostasis, which could contribute to the resistance of FAK inhibition in tumor treatment.Furthermore, our data show that FAK inhibition increases CAFs-released ceramide (CER) and sphingomyelin (SM) levels, whose productions are induced by cellular stress response. 31,32It is possible that FAK inhibitors may function as an exogenous stress to dysregulate the choline homeostasis in stromal cells, due to the low expression of stromal FAK, which mediates no available target for FAK inhibitors exerting their anti-signaling function and subsequent anti-growth effect. 33Overall, dysregulated choline homeostasis and enhanced cellular stress work together to mediate FAK inhibition-induced secretion of PCs from CAFs to mediate the resistance of FAK inhibitor.
Our MS-based phosphoproteomics indicated that AKT2, the stress-induced protein kinase, 34,35 was able to effectively stimulate the production of PCs from CAFs after FAK inhibitor treatment.AKT2 is an important signaling regulator of metabolism and can be stimulated to counteract stress-induced apoptosis. 36Previous study has indicated that stress-responsive FKBP51 activated AKT2 signaling to enhance glucose uptake in skeletal myotubes. 37e identified that FAK inhibition promoted the phosphorylation of the key rate-limiting step enzyme of PC biosynthesis-CCTα at Ser 315/319/323 sites, and then triggered the overproduction of PCs from CAFs.Clearly, the control of CCTs activity is complex and that is involves multiple oncogenic signaling pathways-related factors that modulate expression and function of CCTs. 38,39Using a combination of phosphoproteomics and functional assays, we further discovered that AKT2 interacted with CCTα and induced the phosphorylation of CCTα to improve its activity in stromal cells.When stroma-derived metabolites harshly elevated, tumor cells could quickly utilize these metabolites to boost their own growth and resistance to the cytotoxic effect of chemotherapies. 38,40Importantly, therapeutic strategies by unselectively targeting whole CAFs population are ineffective since the existence of CAFs heterogeneity.We found that CAFs#1 to #4 (AKT2 S128 /CCTα S315/319/323 -positive CAFs) produced the similar effect to induce the ESCC malignancy and impair the antitumor effect of FAK inhibitors in in vitro assays.Thus, we randomly chosen CAFs#1 for further xenograft model and omics assays; in subsequent assays, we have further validated the change of PCs in other CAFs, and confirmed that CAFs #2 to #4 can produce similar biological effects to CAFs #1.ESCC cells have not responded to coculture with CAFs#5 (AKT2 S128 /CCTα S315/319/323 -negative CAFs).We also found that AKT2 S128 /CCTα S315/319/323 -positive CAFs subset provides the adequate supply of PCs for the persistent activation of intratumoral STAT3 maintained by the Tyk2/JAK2 complex, and resultantly induced FAK-targeted therapy resistance and the ESCC malignancy.Thus, targeting stromal AKT2/CCTα axis and theirderived PCs has been suggested as an effective strategy for enhancing the antitumor effect of FAK inhibitor.Specifically, our data also indicate that FAK inhibition-stimulated the activation of AKT2/CCTα axis and PC production uniquely occurred in CAFs, while not in tumor cells.Combination with our previous report that FAK inhibition could effectively inhibit the expression of several metabolism-related molecules and the malignancy of ESCC cells cultured alone.We suggested that identifying the metabolic processes operating in specific CAFs subsets could provide an opportunity for developing novel antitumor strategies.
Hostile microenvironmental conditions within tumors, including nutrient deprivation, oxygen limitation, high metabolic demand, oxidative stress, and drug stimulation, provoke persistent stress to endow malignant cells with greater tumorigenic, metastatic, and drug-resistant capacity. 41Specifically, calcium signaling pathways have been identified to exert important roles in the establishment and maintenance of drug resistance. 42,43Combined these findings with our data, we suggest that rapidly rising Ca 2+ concentration in stroma and increasing the production of stromal metabolites conferred increased resistance to cell death from stress or apoptotic stimuli, providing a drug-resistant stromal niche for TME-mediated tumor malignancy.Thus, the balance between signaling alterations caused by direct effects of FAK inhibition in cancer cells and stromal fibroblasts could potentially be vital to determine the overall treatment outcome.
Drug resistance is the commonly observed issue when targeted therapy is deployed in both preclinical and clinical settings.5][46] In current work, we have uncovered a TME-derived metabolites-induced STAT3 activation in response to FAK inhibition.We offer novel evidence that the crosstalk between TME and tumors is critical for driving the targeted therapy response.Correspondingly, ruxolitinib, baricitinib and S3I-201, targeting different levels of the STAT3 signaling cascade, showed a strong synergism with FAK inhibitor both in vitro and in vivo.Importantly, we have found that FAK inhibitors are unable to exert inhibitory effect on the growth of CAFs, but can facilitate the secretion of PCs from CAFs.Although STAT3 is the important signaling protein in CAFs, and activated STAT3 effectively induces the tumor-promoting function of CAFs, the aim of present study is not to evaluate the tumorpromoting effect of JAKs/STAT3 pathway in CAFs. 24,47,48We have focused on the inhibition of intratumoral FAK and JAK2/STAT3 pathways on the malignant progression of ESCC cells in the presence of CAFs-derived PCs.We will explore the effect of JAKs/ STAT3 signaling pathway on the secretion of PCs or even other lipid metabolites from CAFs.
Present study highlights a previously unclear role of high plasma PCs in facilitating tumor progression and may be exploited as targets for therapeutic development against solid tumors.0][51] In light of our findings, we speculated that high concentration of plasma PCs in ESCC patients play a critical role in ESCC malignancy.Inhibition the effect of PCs on tumor cells can effectively block tumor malignant progression.Taken all together, plasma PCs levels can not only be used as biomarker to discriminate tumor stages but also be utilized as a potential target for tumor treatment or enhancement the antitumor efficacy of targeted therapies.
In conclusion, combining multi-omics, we systematically investigated PCs-based paracrine communication between specific subset of CAFs and tumor cells to limit the antitumor efficacy of FAK inhibitors.Mechanistically, the alteration of CAFs-derived AKT2/CCTα axis and its-activated intratumoral JAK2/STAT3 pathway induces the resistance of FAK inhibitor in tumor treatment.Importantly, PCs can potentially be used as new biomarkers for ESCC diagnosis.These data provide a new strategy for targeting metabolites-related pathway for ESCC treatment (Supplementary Fig. 18).

METHODS AND MATERIALS
Antibodies and reagents All information of antibodies and reagents were listed in Supplementary Table 2.
The siRNA-based approach was applied to generate targeted genes-knockdown cells.Indicated siRNAs were transfected into primary CAFs using Lipofectamine 2000 reagent.For plasmid stable transfection, pcDNA 3.1-Flag plasmid contained AKT2 S128A or CCTα S315/319/323A mutant was transfected into CAFs.Subsequently, positive clones were selected for further experiments.Tansfection efficacy was evaluated using immunoblotting.Sequences of siRNAs were listed in Supplementary Table 3.

Immunoprecipitation (IP) and immunoblotting (IB) analysis
For IP assay, indicated cells were washed with PBS, lysed in NP40 buffer supplemented with protease and phosphatase inhibitors for 30 min, and then centrifuged at 12000 g for 20 min at 4 °C.Supernatants were collected to incubated with indicated primary antibodies (approximately 10 μg antibody/sample) and protein A/ G sepharose beads (ThermoFisher) on a rotator at 4 °C overnight.Then, samples were centrifuged at 4 °C for 5 min at 3000 g, supernatants were discarded, and pellets were washed with 800 μL cold NP40 buffer for 3 times.Finally, beads were collected, and 60 μL loading buffer was added to the beads.The beads were bathed in metal for 5 min, and supernatants were subjected to IB assay.
For IB assay, proteins were separated using sodium dodecyl sulfate (SDS)-PAGE, and transferred onto a nitrocellulose (NC) membrane.After blocking with PBS buffer solution containing 0.1% Tween-20 and 5% nonfat milk for 1 hour, the membranes were incubated with indicated primary antibodies at 4 °C overnight.PBST was used to wash NC membranes for three times.The membranes were incubated with secondary antibodies for 1 h, and then washed an additional three times with PBST and detected by chemiluminescence (ThermoFisher).
Xenograft study Female BALB/c-nu mice (purchased from Beijing Vital River Laboratory) with 3, 4 weeks of age were used in present assay.All animal procedures were approved by Institutional Review Board of Peking University Cancer Hospital & Institute.
For evaluation of lymph node metastasis of ESCC cells, KYSE410 or KYSE510 cells were subcutaneously co-injected with indicated CAFs into the footpads of mice (n = 5/group).The agents used in this assay was consistent with the model that subcutaneous tumor cells inoculation.Treatment was started from week 2 and sustained for 4 weeks.Lymph node volume was evaluated by our reported formula. 12,24CC tissues and IHC staining All procedures and experiments of ESCC tissues were approved by the institutional Review Board of Peking University Cancer Hospital.The protocols of IHC staining and the calculation of staining index were according to our previous studies.12,30 The dilution of primary antibodies was as follow: pFAK Tyr 397 (1:100), pAKT2 Ser 128 (1:100), pCCTα Ser 315/319/323 (1:500), pSTAT3 Tyr 705 (1:4000), or αSMA (1:1500).

Statistical analysis
All data are expressed as the mean ± SD, and statistical analyses are performed by Graphpad software.5][56][57][58][59] For analysis of clinical IHC samples, Chi-square test was used to evaluate the correlation between two factors.Kaplan-Meier method was employed to establish the survival curves of ESCC patients.P-value < 0.05 was considered statistically significant.
Other methods and materials were included in Supplementary file.

DATA AVAILABILITY
The phosphoproteomic data have been deposited in https://www.iprox.cn/page/home.html,and the accession number was: PXD032254.All data in present article are available upon reasonable request from the corresponding authors.

Fig. 2
Fig. 2 Defactinib stimulates PCs secretion from CAFs.a, b CAFs #1 was treated with control or 10 μM defactinib for 24 h, and then CM were collected, and subjected to untargeted metabolomics.The 20 enriched pathways have been shown using bubble chart (a) and bar chart (b).c The defactinib (10 μM) upregulated representative metabolites were shown using heatmap.d The experimental condition of d is consistent with a.The CM from CAFs #1 was subjected to lipidomics.The 10 enriched pathways were shown using bubble chart.Levels of PCs secreted from e 5 cases of CAFs, 4 cases of TAMs, 3 cases of ECs, 2 cases of ESCC and ESCC cell lines-KYSE410 and KYSE510 or f 5 cases of CAFs with/ without 10 μM defactinib or VS4718, were evaluated using quantitative PCs ELISA assay.g, h KYSE410 and KYSE510 cells were treated with 10 μM PC (16:0/20:4) or glycerophospholipid for 4 days, then the growth of indicated ESCC cells was evaluated using MTS assay (g); or for 24 h, the invasion of indicated ESCC cells was evaluated using Transwell assay (h).***P < 0.001.Error bars, mean ± SD of five independent experiments

Fig. 3 Fig. 4
Fig. 3 CAFs-released PCs induce the resistance of defactinib.a KYSE410 and KYSE510 cells were treated with 10 μM PC (16:0/20:4) or glycerophospholipid with defactinib (0-10 μM) for 4 days, then the growth of indicated ESCC cells was evaluated using MTS assay.IC 50 value of defactinib in KYSE410 and KYSE510 cells was shown.b Transwell apparatus with 0.4 μm pore size was used to evaluate the CAFs-derived CCTα or CCTβ-mediated growth of tumor cells in the presence of defactinib.The control siRNA, CCTα siRNA1/2, or CCTβ siRNA1/2 CAFs #1 were plated in the upper chamber of transwell plates.The KYSE410 or KYSE510 cells were respectively plated in the lower chamber of transwell plates, and cocultured with indicated CAFs #1 with defactinib (0-10 μM) for 4 days, and then growth of indicated ESCC cells was measured using MTS assay.IC 50 value of defactinib in KYSE410 and KYSE510 cells was shown.c CAFs #1 were plated in the upper chamber of transwell plates with 0.4 μm pore size.The KYSE410 or KYSE510 cells were respectively plated in the lower chamber of transwell plates, and cocultured with CAFs #1 with miltefosine (25 μM) and defactinib (0-10 μM) for 4 days, and then growth of indicated ESCC cells was measured using MTS assay.IC 50 value of defactinib in KYSE410 and KYSE510 cells was shown.d KYSE410 and KYSE510 cells were treated with 10 μM PC (16:0/20:4) or glycerophospholipid in the presence of defactinib (10 μM) for 24 h, and the invasion of indicated ESCC cells was evaluated using Transwell invasion assay.e Indicated CAFs #1 were cultured in Transwell apparatus with 8 μm pore size, KYSE410 (left panel) or KYSE510 (right panel) cells were cultured in the upper chamber of transwell plates and treated with 10 μM defactinib for 24 h.The invasion of indicated ESCC cells was evaluated using Transwell invasion assay.f CAFs #1 was cultured in Transwell apparatus with 8 μm pore size, KYSE410 or KYSE510 cells were cultured in the upper chamber of transwell plates and treated with 10 μM defactinib with/without miltefosine (25 μM) for 24 h.The invasion of indicated ESCC cells was evaluated using Transwell invasion assay.The invasive ratio of defactinib/respective control was listed.n.s.no significant difference; ***P < 0.001.Error bars, mean ± SD of three to five independent experiments

Fig. 5
Fig.5CAFs-derived AKT2/CCTα axis mediates the resistance of defactinib in ESCC treatment.a Transwell apparatus with 0.4 μm pore size was used to evaluate the growth inhibitory effect of defactinib.The CAFs #1 harbored vector, loss-of-function AKT2 (S128A) or CCTα (S315/319/ 323A) mutant were plated in the upper chamber of transwell plates.The KYSE410 or KYSE510 cells were respectively plated in the lower chamber of transwell plates.After cells were seeded, defactinib (0-10 μM) was added, incubated for 4 days, and then growth of indicated ESCC cells was measured using MTS assay.IC 50 value of defactinib in KYSE410 and KYSE510 cells was shown.b Transwell apparatus with 8 μm pore size was used to evaluate the anti-invasive ability of defactinib (10 μM), the CAFs #1 harbored vector, loss-of-function AKT2 (S128A) or CCTα (S315/319/323A) plasmid were plated in the lower chamber of transwell plates.The KYSE410 or KYSE510 cells were respectively plated in the upper chamber of transwell plates.After cells were seeded, 10 μM defactinib was added, incubated for 24 h, and then tumor invasion was measured using transwell invasion assay.c KYSE410 (upper panel) or KYSE510 (lower panel) cells were respectively coinjected with CAFs #1 harbored vector, loss-of-function AKT2 (S128A) or CCTα (S315/319/323A) mutant into the flank of BALB/c mouse.After the xenografts reached at approximately 80-100 mm 3 .animals were treated with control vehicle or defactinib (25 mg/kg/day, p.o.), as indicated.Tumor volume was measured every 4 days for the indicated period.Curves of tumor volume were listed.After tumors were resected on day 27, the expression of Ki67 (d), CD31 (e) and LYVE1 (f) was assessed using quantitative ELISA assays.g A popliteal lymph node metastasis model was established in mice by inoculating the foot pads with KYSE410 or KYSE510 cells and CAFs #1 harbored vector, loss-of-function AKT2 (S128A) or CCTα (S315/ 319/323A) mutant.After 1 week, mice were treated with control vehicle or defactinib (25 mg/kg/day, p.o.) for 4 weeks.The lymph nodes were enucleated and lymph node volume was calculated.***P < 0.001.Error bars, mean ± SD of three to five independent experiments

Fig. 7
Fig. 7 Stroma-derived AKT2/CCTα axis determines ESCC malignancy in clinical samples.a Representative images for immunohistochemical pAKT2 Ser 128 , pCCTα Ser 315/319/323 , or αSMA in 108 cases ESCC patients.Magnification, 10× as indicated.Percentages of 108 cases ESCC patients with high or low expression of stromal pAKT2 Ser 128 (b) or pCCTα Ser 315/319/323 (c) according to different clinical parameters as follows: tumor stage, tumor status and lymph node status.Two-tailed Pearson χ 2 test.Kaplan-Meier curves of ESCC patients (108 cases) with low vs high expression of stromal pAKT2 Ser 128 (d) or pCCTα Ser 315/319/323 (e).f Stromal pAKT2 Ser 128 or pCCTα Ser 315/319/323 expression associated with intratumoral pStat3 Tyr 705 expression in 108 cases ESCC specimens.Two representative specimens with low and high levels of stromal pAKT2 Ser 128 or pCCTα Ser 315/319/323 were shown.Magnification, 10× as indicated.Two-tailed Pearson χ 2 test.g Representative images for immunohistochemical pFAK Tyr 397 in ESCC patients.Magnification, 10× as indicated After tumor volume reached to approximately 100 mm 3 , animals were treated with defactinib.Defactinib decreased the tumor volumes of KYSE410 or KYSE510 tumor alone (The average tumor volume of KYSE410 or KYSE510 tumor alone at day 27 was 815.36 ± 113.94 or 865.67 ± 148.47 mm 3 ; the average tumor volume of KYSE410 or KYSE510 tumor treated with defactinib at day 27 was 305.29 ± 86.64 or 328.35 ± 64.88 mm 3 ), whereas could not hinder the tumor growth of these two ESCC tumors in the presence of CAFs #1 (The average tumor volume of KYSE410 or KYSE510 tumor in the presence of CAFs #1 at day 27 was 1308.65 ± 213.37 or 1272.19 ± 186.17 mm 3 ; the average tumor volume of KYSE410 or KYSE510 tumor in the presence of CAFs #1 treated with defactinib at day 27 was 1115.25 ± 175.49 or 1106.21 ± 172.5 mm 3 ) (Fig.